The ICP4 protein of Herpes Simplex Virus type 1 (HSV-1) is essential for virus growth, and its known functions are to activate the transcription of most HSV genes and repress the transcription of a few genes known to date. It executes these functions by binding to the genome and interacting with a number of cellular complexes involved in transcription, including TFIID, and the mediator complex. These functions are carried out by ICP4 expressed very early in infection. However, ICP4 is also packaged in the virion tegument. Therefore, the possibility that ICP4 delivered by the virus itself contributes to the transcription of viral genes in vitro, or in neurons in vivo, cannot be ruled out. In this scenario, ICP4 would function in a manner similar to VP16. However, ICP4 is a large and structurally complex protein, and despite our past comprehensive analyses of ICP4-interacting partners in the nucleus, it remains possible that ICP4 interacts with viral and cellular proteins in the cytoplasm that have nothing to do with transcription, and that it functions in other as yet unknown processes in viral infection. In this proposal, we present preliminary data showing that is possible to make infectious virus that does not contain ICP4 in the virion. We also show that ICP4 delivered by the virus particle associates with viral DNA as it does when ICP4 is expressed by the virus. These two observations along with the biological and technical tools available to our laboratory will allow us to determine the involvement of virion-associated ICP4 (vICP4) in infection. In this exploratory grant, we will test several viable hypotheses regarding the function of vICP4 in gene expression, innate immunity, and transport to the nucleus. Three aims are proposed to test aspects of our hypotheses; 1. determine the composition of virions with and without vICP4, 2. determine the fate and consequences of vICP4, and 3. characterize the parameters of infection of viruses with and without vICP4 in the mouse ocular model. Completion of the aims of this exploratory study will enable future more focused studies on the mechanism(s) underlying the function of vICP4, its role in pathogenesis, and the potential use of mutants lacking virion ICP4 as replication competent vaccines or vaccine vectors. !